1. Field of the Invention
The present invention relates to the field of viral proteins, particularly those proteins involved in HIV gene regulation and mutants thereof. The gene which encodes the viral factor is also related to the field of the present invention. Recombinant vectors and host cells including a gene of interest, such as the gene for the viral nucleic acid binding factor is also related to the present disclosure. The present invention also relates to the field of methods for regulating the expression of cellular and viral genes, particularly HIV gene expression, and to methods of treatment, and therapeutic agents for treating acquired immunodeficiency disease and other HIV related diseases or symptoms incident an HIV or AIDS infection. The invention further relates to TAR mutants and cell lines infected with TAR mutants, as well as methods for preparing and producing mutant TAR virus.
2. Background of the Related Art
The regulation of HIV-1 gene expression is dependent on multiple cis-acting control elements in the long terminal repeat (Gaynor, R., 1992). Both DNA and RNA elements in the HIV-1 LTR serve as binding sites for cellular factors. In addition, viral regulatory proteins such as Tat and Rev are involved in the activation of gene HIV-1 expression. The mechanisms by which cellular factors interact with Tat and Rev to increase HIV-1 gene expression are not understood.
The human immunodeficiency virus (HIV) is the causative agent of AIDS (Berkhout et al., 1989; Roy et al., 1990b). In common with other retroviruses (Friedman et al., 1988), HIV contains two long terminal repeats (LTRs) and three conserved genes, gag, pol, and env. It also contains a number of critical regulatory genes including tat and rev which, in conjunction with cellular polymerases and transcription factors, are necessary for the activation of viral gene expression (Feng et al., 1988; Gorman et al., 1982; Pearson et al. 1990). Once HIV-1 integrates into the host cell genome, its gene expression is regulated by cellular transcription factors in a manner similar to that of endogenous cellular genes (Friedman et al., 1988). Unlike cellular genes, unique features in the DNA and RNA regulatory regions of the HIV-1 LTR make it a target for the viral transactivator protein, Tat. The activities of several of the cellular transcription factors which bind to the HIV-1 LTR are altered by parameters such as activation or differentiation of lymphocytes or macrophages, the action of lymphokines, and alterations of signal transduction pathways (Garcia et al., 1988, Weeks et al., 1990). Thus, HIV is subject to many of the same regulatory signals that are important in controlling cellular gene expression.
A variety of viral transactivators including the adenovirus E1A, cytomegalovirus immediate early, and the human T-cell leukemia virus tax proteins are able to activate HIV-1 gene expression (Dingwall et al., 1990; Sodroski et al., 1985). These proteins activate HIV-1 through different regulatory elements including the TATA box, USF binding site, and NF-kB sites respectively (Dingwall et al., 1990; Sodroski et al., 1985). In contrast to these viral transactivator proteins whose activity is relatively permissive, activation by the Tat protein is specific for HIV. Disruption of the tat gene prevents viral replication indicating its essential role in the HIV-1 life cycle (Gorman et al., 1982; Pearson et al., 1990). The Tat protein is known to possess at least three functional domains (Sadaie et al., 1988). These include an amino-terminal activation domain, a cysteine-rich domain which may function in dimerization, and a basic domain which is important in nuclear localization and RNA binding (Fisher et al., 1986; Frankel et al., 1988; Garcia et al., 1988; Harrich et al., 1990; Hauber et al., 1987; Sadaie et al., 1988). Despite knowledge of these details, the mechanism of Tat activation remains open to question, and thus an identification of potential interactions between Tat and cellular factors is important for understanding Tat function.
A number of cis-acting regulatory elements in the HIV-1 LTR are critical for basal and Tat-induced gene expression. These include the enhancer, SP1 (Gentz et al.; Modesti et al., 1991), TATA (Sadaie et al., 1988; Selby et al., 1990; Marciniak et al., 1990), and TAR elements (Brake et al., 1990; Malim et al., 1989; Selby et al., 1990; Templeton, 1992). Each of these elements serves as a binding site for cellular transcription factors. Though the SP1 and TATA elements influence the basal level of HIV-1 gene expression, they also play a role in determining the level of activation by the transactivator protein Tat (Modesti et al., 1991). The TAR element which forms a stable stem-loop RNA structure extending from +1 to +60 is critical for Tat activation (Brake et al., 1990; Malim et al., 1989; Rosen et al., 1985; Templeton 1992). A number of studies using fusions of Tat to other known DNA or RNA binding proteins indicate that Tat is able to activate HIV-1 gene expression when bound to either DNA or RNA (Mann et al., 1991; Modesti et al., 1991). Thus it is likely that factors binding to both DNA and RNA regulatory elements influence the degree of Tat activation.
One HIV-1 regulatory element, TAR, is critical for tat activation (Rosen et al., 1985). TAR forms a stable stem loop RNA structure (Lu et al., 1993; Okamoto and Wong-Staal, 1986) that contains three critical elements. These include a three nucleotide bulge between +23 and +25 (Berkhout and Jeang, 1989; Calnan et al., 1991; Dingwall et al., 1990; Roy et al., Genes & Dev., 1990; Roy et al., J. Virol., 1990), a six nucleotide loop between +30 and +35 (Berkhout and Jeang, 1989; Feng and Holland, 1988; Garcia et al., 1989; Roy et al., Genes & Dev., 1990; Roy et al., J. Virol., 1990; Wu et al., 1991), and the upper stem structure between +18 and +43 (Feng and Holland, 1988; Garcia et al., 1989; Hauber and Cullen, 1988; Jakobovits et al., 1988; Roy et al., J. Virol., 1990; Selby et al., 1989). Tat binds directly to the TAR RNA bulge (Calnan et al., 1991; Dingwall et al., 1989; Dingwall et al., 1990; Roy et al., Genes & Dev., 1990; Weeks and Crothers, 1991) while a cellular factor designated TRP-185/TRP-1 binds to the loop sequences (Sheline et al., 1991; Roy et al., J. Virol., 1990) and other proteins have been found to bind to the stem (Gatignol et al., 1991). In addition, TAR DNA serves as the binding site for a variety of proteins (Garcia et al., 1987; Jones et al., 1988; Kato et al., 1991; Wu et al., 1988) which may be involved in the generation of short transcripts from the HIV-1 LTR (Ratnasabapathy et al., 1990; Sheldon et al., 1993). Heterologous constructs containing TAR fused to a variety of different promoters are capable of being activated by tat indicating the critical role of this element (Berkhout et al., 1992; Muesing et al., 1987; Peterlin et al., 1986; Ratnasabapathy et al., 1990; Wright et al., 1986). Thus TAR is a complex regulatory element which is important in modulating tat-mediated gene expression from the HIV-1 LTR.
The function of the TAR element has been studied by transient expression assays of wild-type and mutant HIV-1 LTR templates in both the presence and absence of tat (Berkhout and Jeang, 1989; Feng and Holland, 1988; Garcia et al., 1989; Hauber and Cullen, 1988; Jakobovits et al., 1988; Rosen et al., 1985; Roy et al., J. Virol., 1990; Selby et al., 1989). In addition, transient transfection assays of wild-type and TAR mutant proviral constructs have also been used to demonstrate a critical role for TAR in regulating HIV-1 gene expression (Hauber and Cullen, 1988). However, it has not previously been possible to generate high titer stocks of TAR mutant viruses to study the effects of these mutations on gene expression and growth properties. Viral mutants in other HIV-1 regulatory elements such as NF-.kappa.B and SP1 have been constructed and the effects of these mutations on viral growth properties have been analyzed (Leonard et al., 1989; Lu et al., 1989; Parrott et al., 1991; Ross et al., 1991). These studies indicated that viral growth properties were affected by both the specific mutation introduced and the cell type on which viral growth was analyzed.
Mutagenesis has localized a region of TAR RNA between +18 and +44 as an essential element for activation by Tat (Brake et al., 1990; Calnan et al., 1991; Dayton et al., 1986; Glen et al., 1987; Malim et al., 1989; Selby et al., 1990; Elroy-Stein et al., 1989). Several elements in this RNA region including the bulge (+23/+25), the loop (+30/+35), and the stem structure are required for complete Tat-activation (Brake et al., 1990; Dayton et al., 1986; Glen et al., 1987; Malim et al., 1989; Selby et al., 1990; Elroy-Stein et al., 1989). The function of the stem structure is likely to maintain the position of the bulge and loop structures. The bulge region in TAR RNA serves as the binding site for Tat though the loop sequences also influence Tat binding (Fisher et al., 1986; Frankel et al., 1988; Garcia et al., 1988; Hauber et al., 1987). In addition, cellular factors are also capable of binding to the bulge sequences. The interaction between Tat and the TAR RNA bulge is very specific in that a change of one nucleotide at +23 in the bulge is sufficient to disrupt Tat binding (Fisher et al., 1986; Frankel et al., 1988; Garcia et al., 1988; Hauber et al., 1987). The basic domain of Tat is necessary and sufficient for binding to the TAR RNA bulge (Fisher et al., 1986; Frankel et al., 1988; Garcia et al., 1988; Hauber et al., 1987). Extensive mutagenesis of the Tat protein indicates that arginine residues at positions 52 and 53 of Tat are especially critical for interacting with phosphate groups in the TAR RNA bulge (Frankel et al., 1988). The Tat binding to the TAR RNA bulge is thus highly specific and of great affinity.
In contrast to the bulge which binds a viral protein, the loop sequences serve as a binding site for cellular factors that may cooperate with Tat in activating HIV-1 gene expression (Selby et al., 1989; Siomi et al., 1990). Fractionation of HeLa nuclear extract and gel retardation and UV crosslinking using TAR RNA probes indicate that two different cellular proteins p68 and TRP-185 bind to the TAR RNA loop sequences. TRP-185 is a ubiquitously expressed 185 kDa protein whose binding to TAR RNA is regulated by additional cofactor proteins. These cofactors likely function by post-translational modification of TRP-185 i.e., phosphorylation. TRP-185 binding to TAR RNA requires wild-type loop sequences and an intact bulge structure. The binding of TRP-185 to TAR RNA, unlike that of Tat, is not markedly influenced by the primary sequences of the bulge region. Both Tat and TRP-185 activate HIV-1 LTR gene expression in in vitro transcription assays, but whether these proteins directly interact is not known. These results indicate that Tat activation via the TAR element may require interactions between Tat and cellular transcription factors.
Activation of the HIV-1 LTR by Tat proteins with an altered basic domain has previously been demonstrated to be strongly dependent on the concentration of transfected DNA (Ruben et al., 1989; Hauber et al., 1989). However, how this finding relates to the overall activation of the HIV-1 LTR remains to be determined.
It is critical to determine how Tat modulates the transcriptional apparatus to increase HIV-1 gene expression. Tat stimulates steady state RNA levels synthesized from the HIV-1 LTR approximately 20 to 50-fold. Nuclear run-on experiments using the HIV-1 LTR indicate that Tat stimulates transcriptional initiation. However another effect of Tat function is seen when nascent RNA is measured at various positions downstream of the HIV-1 LTR initiation site in both the presence and absence of Tat (Jones et al., 1986). Though several studies demonstrate an increased number of RNA molecules synthesized from proximal portions of the HIV-1 LTR (near the initiation site) in the presence of Tat, the predominant effect of Tat appears to be a marked increase in the level of RNA synthesized at promoter distal sites (between 500 to 1000 nucleotides from the initiation site) (Jones et al., 1986). In vitro analysis of Tat transactivation also supports an effect on transcriptional elongation. The ability of Tat to increase the number of elongated transcripts may be one explanation for the decrease in the number of short transcripts which are synthesized from the HIV-1 LTR in the absence of Tat. These short transcripts terminate around +60 in the TAR element and may reflect the products of poorly processive transcription complexes. Thus, Tat may function at multiple steps in the transcriptional pathway to increase both the initiation and elongation of transcripts from the HIV LTR.
Mutations in a number of HIV-1 genes including tat (Pearson et al. (1990)), rev ((Malin et al. (1989)), and gag (Trono et al. (1989)) result in proteins with a dominant negative or transdominant phenotype that interfere with the function of the corresponding wild-type proteins. Recently, a .DELTA.tat mutation has been described by the present inventors. The .DELTA.tat mutant gene therein encoded a 54 amino acid length HIV protein having truncated basic domain (Pearson et al. (1990)). The "basic domain" of the tat gene includes 9 amino acids and is defined by amino acid residues 49-57 of the first 72 amino acids encoding the Tat protein (Pearson et al. (1990)). Three (3) of the amino acid residues of the basic domain of the HIV Tat protein were eliminated in the .DELTA.tat to provide the final protein product, leaving six (6) of the residues of the basic domain unchanged. While the .DELTA.tat-encoded protein was found to inhibit Tat activation of the HIV-1 LTR when the vector expressing it was present in a 5- to 30-fold molar excess over a vector expressing the wild-type Tat, the mutations were not found to result in a transdominant phenotype.
Further characterization of the precise mechanisms controlling HIV gene expression in regard to the role of the "basic domain" of the tat gene has not been explored, despite the impact such would have in providing more potent and effective therapeutic agents for treating HIV infections.
Previous data have demonstrated that Tat protein is capable of entering cells in culture when added to the tissue culture media (Rice et al., 1990). Though the mechanism of entry is not understood it appears to be a result of endocytosis. To develop transdominant Tat mutant peptides for potential therapeutic use it would be important to develop transdominant mutants of minimal size. This is due to the fact that the amount of partial products and the yield of peptides decrease significantly as their size is increased. A construct which encoded a peptide capable of providing defective activation of HIV LTR gene expression and an ability to antagonize wild-type Tat function, and which was of sufficiently small size to optimize partial product and peptide yield would enable the production of an entirely new class of therapeutic agents used in the treatment and potential cure of HIV infections.
A number of elements in the HIV-1 LTR are critical for the regulation of gene expression. Previous studies have revealed that the enhancer, SP1, TATA and TAR regions are all critical for both basal and tat-induced gene expression (Gaynor, R., 1992). Mutations in the enhancer, SP1, and TATA elements have been inserted into HIV-1 proviral constructs and their effects on gene expression and viral growth assayed (Harrich et al., 1989; Leonard et al., 1989; Lu et al., 1993; Parrott et al., 1991; Ross et al., 1991). These studies indicated that both the specific regulatory element which was mutated and the cell-type which was infected were determinants of the level of viral gene expression. Mutations of some regulatory elements such as the enhancer have very different effects on viral growth than seen with transient assays. Mutation of NF-.kappa.B motifs are very deleterious to HIV-1 gene expression when assayed by transient expression (Nabel et al. 1987), but viruses containing these same mutations exhibit only slight decreases in viral growth properties (Leonard et al. 1989; Ross et al., 1991). Thus it is critical to determine how mutations of different HIV-1 regulatory regions alter gene expression following both transient assays and in the context of virus. Since TAR is critical for tat-activation, studies of viruses containing mutations in this regulatory element are critical for a better understanding of the factors controlling HIV-1 gene expression. It has not been possible, before the present invention, to assay the effects of TAR mutations on viral growth and gene expression because of the inability to generate such proviruses.